DESCRIPTION: Antibody therapeutics are a rapidly growing class of glycoprotein pharmaceuticals. Many antibody drugs bind to receptors on cells and either initiate or accelerate cell death and depletion. Several antibody drugs are nearing the end of patent protection and the efficacy and safety of biosimilars, which are the generic replacements, must be established. Although glycans comprise only ~3% of the mass of an antibody drug, glycosylation significantly impacts the effect the antibody has on stimulating the immune system to destroy specific cells. There is a critical need to profile antibody glycosylation, but the analysis of glycans is challenging. This is because glycans are defined by the variation in the type of monomeric saccharide unit, the position of the linkage between adjacent saccharide monomers, and chain branching. The proposed research generates a phospholipid-based enzyme mobility shift assay to rapidly sequence antibody glycosylation and establish both the composition and linkage orientation of glycan monomers that are implicated in antibody function. Two analytical strategies support a systematic approach to rapidly sequence glycans. Exoglycosidase enzymes that cleave only the terminal monomers with high specificity are integrated into a microscale separation channel. The glycan is electrophoretically driven into the enzyme, incubated for several minutes and then separated in the same channel using electrophoresis. When the terminal monomer of a glycan matches the specificity of the enzyme it is cleaved from the glycan. This decreases the charge-to-size ratio of the glycan and results in a shift in migration time that is used to identify both the monomer and the linkage. Aim 1 activities improve the characterization of antibodies by integrating nanoliter volumes of enzymes in a programmable capillary electrophoresis instrument. Glycans are subject to sequencing with a series of enzymes. This automated method can assay femtomolar glycans and consumes only a few nanoliters of enzyme for each incubation. Aim 2 activities dramatically increase the throughput of the approach by performing multiple exoglycosidase reactions simultaneously. This is accomplished in microfluidic devices with parallel channels or channel-free separations. The heart of this microscale sequencing is a unique phospholipid separation additive that is a thermally-responsive material with low viscosity at 25C, and gel- like viscosity at 30C. These properties make it easier and more practical to perform microscale sequencing in capillary separations with an automated instrument or parallel microfluidic device.